Isolation of long-lived pluripotent hematopoietic stem cells is of cardinal importance to hematopoietic stem cell biology as well as gene therapy of stem cells. In collaboration with P. Goodell, we have demonstrated a novel population of hematopoietic cells based on vital staining with Hoechst 33342. This minor population (0.01 - 0.03% of bone marrow mononuclear cells in rhesus) exhibits a characteristic uptake of the dye Hoechst 33342 and is referred to as the side population. Murine studies demonstrated that the side population is highly enriched for bone marrow long-term repopulating cells. In the rhesus we have shown that this population is largely CD34-, although heterogeneous with respect to the surface expression of lineage associated epitopes. Side population cells do not give rise to myeloid or erythroid clonogenic progenitors when cultured in methylcellulose. Side population cells demonstrate a marked enrichment for long term culture initiating cells (LTCIC), a measure of primitive hematopoietic cells. Using a limiting dilution LTCIC, we observed a LTCIC frequency of 1 in 3 for the side population, compared to 1 in 75-85 for CD34+ cells. We are currently examining